EP0449812B1 - Method of pre-treating samples in peroxidase-catalyzed enzyme assays - Google Patents

Method of pre-treating samples in peroxidase-catalyzed enzyme assays Download PDF

Info

Publication number
EP0449812B1
EP0449812B1 EP89903308A EP89903308A EP0449812B1 EP 0449812 B1 EP0449812 B1 EP 0449812B1 EP 89903308 A EP89903308 A EP 89903308A EP 89903308 A EP89903308 A EP 89903308A EP 0449812 B1 EP0449812 B1 EP 0449812B1
Authority
EP
European Patent Office
Prior art keywords
assay
peroxidase
analyte
blood
oxidizing agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP89903308A
Other languages
German (de)
English (en)
French (fr)
Other versions
EP0449812A4 (en
EP0449812A1 (en
Inventor
Patrick A. Mach
Jeffrey A. Thompson
Richard S. Creager
Cheri W. Fink
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sanofi Diagnostics Pasteur Inc
Original Assignee
Sanofi Diagnostics Pasteur Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sanofi Diagnostics Pasteur Inc filed Critical Sanofi Diagnostics Pasteur Inc
Publication of EP0449812A1 publication Critical patent/EP0449812A1/en
Publication of EP0449812A4 publication Critical patent/EP0449812A4/en
Application granted granted Critical
Publication of EP0449812B1 publication Critical patent/EP0449812B1/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/825—Pretreatment for removal of interfering factors from sample
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25125—Digestion or removing interfering materials

Definitions

  • This invention relates to an oxidative sample treatment to eliminate interference of blood or blood products in enzyme assays.
  • US-A-4444880 describes an immunoassay employing a bibulous support to which is attached a mip and a mip-peroxidase conjugate which binds to the mip on a support where the peroxidase catalyses the formation of a dye which binds to said support and ascorbate in said sample interferes with the production of said dye, and wherein the support is impregnated with a sufficient amount of periodate to reduce said ascorbate interference to a level which does not interfere with the detection of the analyte.
  • Chemical Abstracts, 101 (1984): 207202s describes a method and sampling apparatus by which a biological sample solution is adsorbed on a carrier containing an oxidizing agent to eliminate interference, the sample is redissolved in a buffer, and the components of interest are detected. Blood serum or urine was adsorbed on the porous support containing the oxidizing agent.
  • Chemical Abstracts, 104 (1986): 31375w discloses a container for sample analysis based on an enzymic and diazo coupling reactions, wherein the inner surface is treated with a H2O-solution oxidizing agent to avoid interference by reducing substances.
  • a glucose test strip was dipped into a urine sample in the cup. Compared to controls, interference by ascorbic acid was minimal.
  • enzyme immunoassays EIAs
  • an assay reagent is labeled with an enzyme, the reagent becoming bound to a solid support in an amount that depends upon the amount of analyte in the sample.
  • Enzyme substrate generally added in a final step of the assay, reacts with the enzyme to generate a detectable signal which is related to the amount of analyte present in the sample.
  • Blood or blood products are common sources of assay interference in enzyme assays which employ peroxidase as the enzyme label.
  • the blood or blood products help convert the enzyme substrate to its oxidized products, giving rise to positive assay interference.
  • the heme component of hemoglobin can bind to solid supports used in EIAs to immobilize reagents.
  • the heme moiety also called "microperoxidase" present during signal development in peroxidase-catalyzed EIAs generates non-specific signal (i.e. color development) and is a source of positive interference.
  • a method for detecting the presence of an analyte using a peroxidase-catalysed enzyme assay comprises the steps of:
  • a kit for performing a diagnostic enzyme immunoassay to detect the presence of an analyte in a clinical specimen is characterized by
  • the methods of this invention can be used with patient specimens obtained by conventional methods, such as swabs, washes, aspirates and the like, taken from the eye, the nares at the back of the nose, cervix, vagina, urethra, rectum, throat, blood, serum, plasma and the like.
  • the oxidizing agent may be added directly to the patient sample.
  • a solution of oxidizing agent may be added to a patient sample just prior to taking an aliquot of the sample for use in the assay.
  • the oxidizing agent solution may be added to a reagent, such as a detergent solution, that is used to extract the patient sample from a swab or the like.
  • a reagent such as a detergent solution
  • This extraction solution is added to a tube containing a swab sample, mixed, and allowed to incubate for a predetermined length of time, followed by expulsion of liquid from the swab.
  • the liquid containing the patient sample is then evaluated using an EIA or other enzyme assay such as a peroxidase-catalyzed enzyme assay.
  • a solution of oxidizing agent is added to the liquid extracted from a patient swab using a detergent or the like. This solution of oxidizing agent and patient sample is allowed to incubate for a predetermined length of time, following which an appropriate aliquot is taken for use in the enzyme assay.
  • the method of this invention may be used in any enzyme assay but is particularly useful in a passive or active, capture-dependent, peroxidase-catalyzed, sandwich-type EIA.
  • the sandwich-type EIA is preferably one in which an antibody:antigen:antibody sandwich tethered to a solid support is formed.
  • antigen or antibody is bound to a solid support such as filter paper, test tubes made from polyethylene, polystyrene, polypropylene or other suitable materials, latex particles, glass or plastic beads, magnetic particles or the like.
  • Patient samples are contacted with the solid support and the specific analyte being assayed binds to the anchored ligand on the solid support.
  • a second antibody with specificity to the analyte and directly or indirectly labeled with peroxidase is added and, after the reaction mixture has been allowed to incubate for a sufficient time to allow the reaction to occur, the solid support is washed. If the second antibody is directly labeled, then, after the solid support is washed, enzyme substrate is added to the solid support and the resulting signal is measured by colorimetric or spectrophotometric techniques. If the second antibody is unlabeled, then a labeled antiglobulin directed against the second antibody may be added and the solution allowed to incubate for a predetermined time before washing and determination of the quantity of label by conventional techniques.
  • ligand-receptor pair refers to a pair of compounds of which one, a "receptor” is capable of recognizing a particular spatial and polar organization of the other ("ligand") or portion thereof, and is capable of binding to that compound.
  • illustrative receptors forming the other half of a ligand-receptor pair include antibodies, enzymes, lectins, Fab fragments, complementary nucleic acids and the like.
  • the invention is useful in detecting a broad range of analytes.
  • U.S. Patent 4,374,925 and U.S. Patent 3.817,837 set out excellent lists of analytes which are part of specific binding pairs.
  • Other examples of binding pairs include nucleic acids and complementary nucleic acids.
  • Analytes of particular interest include viruses, bacteria, fungi and protozoans, specific products and assemblages thereof and macromolecules and products of living organisms.
  • Antibodies useful with enzyme assays may be raised in humans or non-human species such as guinea pig, goat, horse, rabbit, sheep, etc. by immunization with appropriate antigens in accordance with known methods. Monoclonal antibody to appropriate antigens may also be used with the methods of this invention.
  • Assay interference can be detected by comparing assay results with results obtained using other evaluation methods, i.e., culture techniques, microscopic analysis, and the like.
  • a specific example of the interference by blood or blood products of a direct antigen EIA is described as follows in connection with an assay for the detection of Chlamydia trachomatis . Samples for the detection of chlamydia were taken from perspective patients. Direct fluorescent labeling techniques were used to determine whether chlamydia was present in a sample, as follows: Antibody that binds specifically to chlamydia and that was labeled with a fluorescent agent was obtained. Labeled antibody of this type is commercially available.
  • a volume of specimen extract was obtained and centrifuged to form a pellet comprised of chlamydial organisms and debris from the sample.
  • the pellet was resuspended in a minimal volume of buffer, spotted on a microscope slide, fixed with methanol and stained with the labeled antibody reagent.
  • the antibody bound to the chlamydia, if any, on the slide.
  • the slide was examined using an appropriate microscope to determine whether chlamydia were present.
  • samples not treated with the oxidizing agent prior to performing the EIA a significant number of specimens that tested negative with the direct fluorescent method yielded positive EIA results in the absence of the additives described in this invention.
  • an appropriate concentration of hydrogen peroxide was diluted into the buffered detergent solution used for treating patient samples.
  • the patient swab samples were first treated with 0.5 ml of sample treatment solution without hydrogen peroxide. After removal of swab the remaining specimen was divided into two equal volumes. To one of the aliquots was added a sample treatment solution with hydrogen peroxide, to the other an equal volume of sample without hydrogen peroxide was added. The results are shown in Table 2.
  • the oxidizing agents effective in the method of this invention include sodium hypochlorite, sodium meta-periodate, and hydrogen peroxide and other oxidizing agents that are sufficiently reactive with blood or blood products to substantially eliminate interference in a peroxidase-catalyzed, enzyme assay caused thereby.
  • concentration of oxidants added to the sample may be widely varied so long as it exceeds a particular minimum. Usually a solution of at least about 0.3% volume/volume ("v/v”) of hydrogen peroxide can be used. Amounts in excess of 1.0% v/v of hydrogen peroxide are usually not required.
  • 0.1 ml of 3.0% v/v hydrogen peroxide solution was placed in a 12 x 75 mm test tube.
  • a urogenital swab specimen was placed into the test tube and extracted by adding to the test tube 1.0 ml of a sample treatment solution containing 0.2M Tris (hydroxymethyl)-amino- methane hydrochloride ("Tris HCl") at pH 8.0, 0.1M sodium chloride, 0.005M disodium ethylene diamine tetraacetic acid ( Na2EDTA) and 0.05% weight/volume 3-[(3-cholamido-propyl)-diamethyl ammonio]-1-propane sulfonate (CHAPS)) to the test tube.
  • Tris HCl Tris (hydroxymethyl)-amino- methane hydrochloride
  • Na2EDTA disodium ethylene diamine tetraacetic acid
  • CHAPS weight/volume 3-[(3-cholamido-propyl)-
  • the swab was allowed to incubate in the sample treatment solution for at least 10 minutes.
  • the tube was vortexed for 30 seconds.
  • the swab was expressed against the side of the tube, discarded and an appropriate volume of the sample was removed from the tube for use in a chlamydia specific EIA as described in Example 4.
  • a urogenital swab specimen was placed in a 12 x 75 mm glass test tube.
  • a 1.0 ml volume of sample treatment solution (see example 1) containing 0.3% v/v hydrogen peroxide was added to the tube to extract the sample from the swab.
  • the swab was allowed to incubate in the above-described solution for at least ten minutes, vortexed, wrung out against the side of the tube to expel absorbed liquid and discarded. An appropriate volume of sample was removed from the tube for assay in a chlamydia specific EIA as described in Example 4.
  • a urogenital swab specimen was placed in a 12 x 75 mm test tube and extracted into 1.0 ml of the sample treatment solution of Example 1.
  • the swab was allowed to incubate in that solution for at least 10 minutes and the tube was then vortexed for about 30 seconds. After the swab was expressed, 0.1 ml of 3.0% v/v hydrogen peroxide was added to the sample and the tube was vortexed for another ten seconds.
  • An appropriate volume of sample was removed from the tube for use in a chlamydia specific EIA as described in Example 4.
  • HRP horseradish peroxidase

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
EP89903308A 1988-12-15 1989-02-16 Method of pre-treating samples in peroxidase-catalyzed enzyme assays Expired - Lifetime EP0449812B1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US284541 1988-12-15
US07/284,541 US4978632A (en) 1988-12-15 1988-12-15 Method of pre-treating samples in peroxidase-catalyzed enzyme assays
PCT/US1989/000633 WO1990007115A1 (en) 1988-12-15 1989-02-16 Method of pre-treating samples in peroxidase-catalyzed enzyme assays

Publications (3)

Publication Number Publication Date
EP0449812A1 EP0449812A1 (en) 1991-10-09
EP0449812A4 EP0449812A4 (en) 1991-10-30
EP0449812B1 true EP0449812B1 (en) 1995-09-13

Family

ID=23090596

Family Applications (1)

Application Number Title Priority Date Filing Date
EP89903308A Expired - Lifetime EP0449812B1 (en) 1988-12-15 1989-02-16 Method of pre-treating samples in peroxidase-catalyzed enzyme assays

Country Status (8)

Country Link
US (1) US4978632A (da)
EP (1) EP0449812B1 (da)
JP (1) JP2869804B2 (da)
AT (1) ATE127922T1 (da)
CA (1) CA1338999C (da)
DE (1) DE68924272T2 (da)
DK (1) DK115091A (da)
WO (1) WO1990007115A1 (da)

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69016870T2 (de) * 1989-08-04 1995-07-20 Kyowa Medex Co Ltd Verfahren und Satz zur Bestimmung von Bestandteilen.
GB2245062B (en) * 1990-06-12 1994-03-16 Nat Res Dev Antioxidant assay
US5413911A (en) * 1990-12-14 1995-05-09 Abbott Laboratories Determination of tricyclic antidepressant drugs in the presence of interfering substances
US5789152A (en) * 1991-04-30 1998-08-04 Basil T. Hone Composition and method for detecting HIV with baculovirus derived vesicles
ATE197504T1 (de) * 1991-12-13 2000-11-11 Abbott Lab Bestimmung trizyklischer antidepressiva in gegenwart störender substanzen
DE4215275A1 (de) * 1992-05-09 1993-11-11 Merck Patent Gmbh Mittel und Verfahren zur Behandlung von Körperflüssigkeiten bei der Bestimmung von Neopterin
US5484706A (en) * 1993-05-19 1996-01-16 Pasteur Sanofi Diagnostics Immunoassay for analytes in samples using alkylating agents
US5494801A (en) * 1993-12-03 1996-02-27 Biostar, Inc. Microorganism antigen extraction methods
US5478729A (en) * 1994-04-28 1995-12-26 Syntex (U.S.A.) Inc. Immunoassay for homocysteine
DE19628484A1 (de) * 1996-07-15 1998-01-22 Boehringer Mannheim Gmbh Blutersatzmittel-Entstörung durch Peroxide
US6265220B1 (en) 1998-06-29 2001-07-24 Edwin F. Ullman Assay for homocysteine
WO2007092360A2 (en) * 2006-02-02 2007-08-16 Ethicon, Inc. Diagnostics and methods for removal and detection of interferents
CA2706763C (en) * 2007-11-27 2013-07-30 Laboratory Corporation Of America Holdings Enhanced method for detecting and/or quantifying an analyte in a sample
US11401654B2 (en) * 2018-03-19 2022-08-02 Softray, Inc. Apparatus and methods for pre-treating swabs prior to collection of specimens to reduce false positive detections
US20190309271A1 (en) 2018-03-20 2019-10-10 Rubius Therapeutics, Inc. Therapeutic cell systems and methods for treating homocystinuria
CN112730853A (zh) * 2020-12-15 2021-04-30 深圳天辰医疗科技有限公司 抑制素b检测试剂盒和抑制素b的检测方法

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5592698A (en) * 1978-12-31 1980-07-14 Kyoto Daiichi Kagaku:Kk Test strip for glucose determination
CA1146852A (en) * 1979-01-31 1983-05-24 Koichi Kondo Reagent for latex agglutination
US4299815A (en) * 1980-02-08 1981-11-10 Hoffmann-La Roche Inc. Carcinoembryonic antigen determination
AU543007B2 (en) * 1980-04-15 1985-03-28 Technicon Instruments Corportion Agglutination immunoassay
IT1134333B (it) * 1980-11-19 1986-08-13 F F A Spa Sa Processo per stabilizzare mediante incapsulamento il fosforo rosso per impiego come ritardante di fiamma dei materiali polimerici e prodotto cosi' ottenuto
US4444880A (en) * 1982-07-27 1984-04-24 Syva Company Periodate removal of ascorbate interference in dipsticks for immunoassays
JPS59122939A (ja) * 1982-12-28 1984-07-16 Matsushita Electric Ind Co Ltd 生体試料液採取用具および試料中の特定成分濃度の測定法
US4587220A (en) * 1983-03-28 1986-05-06 Miles Laboratories, Inc. Ascorbate interference-resistant composition, device and method for the determination of peroxidatively active substances
JPS60198460A (ja) * 1984-03-22 1985-10-07 Terumo Corp 試験容器
US4766065A (en) * 1984-08-23 1988-08-23 Becton Dickinson And Company Detection of cell membrane protein
US4810630A (en) * 1987-03-30 1989-03-07 E. I. Du Pont De Nemours And Company Use of polyoxyethylene ethers to improve the performance of immunoassays employing peroxidase conjugates
WO1990002202A1 (en) * 1988-08-16 1990-03-08 Cetus Corporation Reduction of peroxidatic and catalatic interference with assays of peroxidatic activity

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 101, no. 23, 03 December 1984, Columbus, OH (US); p. 320, no. 207202s# *
CHEMICAL ABSTRACTS, vol. 104. no. 5, 03 February 1986, Columbus, OH (US); p.296, no. 31375w# *
CHEMICAL ABSTRACTS, vol. 98, no. 3, 17 January 1983, Columbus, OH (US); M.W. McGOWAN et al., p. 278, no. 13878r# *

Also Published As

Publication number Publication date
WO1990007115A1 (en) 1990-06-28
DE68924272T2 (de) 1996-03-21
EP0449812A4 (en) 1991-10-30
CA1338999C (en) 1997-03-18
EP0449812A1 (en) 1991-10-09
JP2869804B2 (ja) 1999-03-10
JPH05508006A (ja) 1993-11-11
DE68924272D1 (de) 1995-10-19
ATE127922T1 (de) 1995-09-15
DK115091A (da) 1991-08-12
US4978632A (en) 1990-12-18
DK115091D0 (da) 1991-06-14

Similar Documents

Publication Publication Date Title
EP0449812B1 (en) Method of pre-treating samples in peroxidase-catalyzed enzyme assays
EP0171150B1 (en) Method and apparatus for assaying with optional reagent quality control
JP3009155B2 (ja) 免疫クロマトグラフイー分析方法
US5126276A (en) Method for the determination and measurements of more than one unknown material in a single surface of a multianalytic assay
US5024935A (en) Dye-providing composition, diagnostic test kit and their use in method for ligand determination using peroxidase labeled-receptor
EP0402396B1 (en) Chlamydia assay employing base treatment
US5063151A (en) Immunoassay method and kit
US5366864A (en) Buffered wash composition, insolubilizing composition, test kits and method of use
US7015005B2 (en) Methods and devices for detecting non-complexed prostate specific antigen
CA1285224C (en) Simultaneous extraction of a ligand from a sample and capture by anti-ligands therefor in ligand/anti-ligand assays
CZ83393A3 (en) Process of treating humors when determining neopterin and means for carrying out said process
EP1192467B1 (en) Chemiluminescence redox assay to quantify analytes in biological samples
JP3228791B2 (ja) 検体中の抗原又は抗体の測定法
JPH0736015B2 (ja) 診断試験キットおよびリガンドの測定方法
EP0348144B1 (en) Method for treating mucus-containing immunoassay samples
CN113493514B (zh) 抗三碘甲状腺原氨酸单抗的酶结合物、总三碘甲状腺原氨酸定量检测试剂盒及其使用方法
WO1990002336A1 (en) Immunoassay for determining a chlamydial antigen comprising pretreatment of the sample with a chelating agent
EP0399127A1 (en) Homogeneous immunochemical method for determining haptens by means of ion selective electrodes
JPH08509064A (ja) 化学的に架橋したタンパクの固相支持体上の固定化
JPH0695956B2 (ja) ペルオキシダーゼ活性の高感度検出
Monroe Enzyme Channelling Immunoassay (ECIA): A Unique and Rapid Quantitative Technique
JPH11258238A (ja) 免疫学的凝集測定用試薬キット

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19910614

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

A4 Supplementary search report drawn up and despatched

Effective date: 19910910

AK Designated contracting states

Kind code of ref document: A4

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

17Q First examination report despatched

Effective date: 19931108

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: SANOFI DIAGNOSTICS PATEUR, INC.

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Effective date: 19950913

Ref country code: AT

Effective date: 19950913

REF Corresponds to:

Ref document number: 127922

Country of ref document: AT

Date of ref document: 19950915

Kind code of ref document: T

REF Corresponds to:

Ref document number: 68924272

Country of ref document: DE

Date of ref document: 19951019

ET Fr: translation filed
ITF It: translation for a ep patent filed
PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 19960229

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed
REG Reference to a national code

Ref country code: GB

Ref legal event code: IF02

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20050203

Year of fee payment: 17

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SE

Payment date: 20050204

Year of fee payment: 17

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20050208

Year of fee payment: 17

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20050210

Year of fee payment: 17

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES;WARNING: LAPSES OF ITALIAN PATENTS WITH EFFECTIVE DATE BEFORE 2007 MAY HAVE OCCURRED AT ANY TIME BEFORE 2007. THE CORRECT EFFECTIVE DATE MAY BE DIFFERENT FROM THE ONE RECORDED.

Effective date: 20050216

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 20050216

Year of fee payment: 17

Ref country code: GB

Payment date: 20050216

Year of fee payment: 17

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20060216

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20060217

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20060228

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20060228

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20060901

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20060901

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

EUG Se: european patent has lapsed
GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20060216

NLV4 Nl: lapsed or anulled due to non-payment of the annual fee

Effective date: 20060901

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20061031

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20060228

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IT

Payment date: 20060228

Year of fee payment: 18

PGRI Patent reinstated in contracting state [announced from national office to epo]

Ref country code: IT

Effective date: 20080301

PGRI Patent reinstated in contracting state [announced from national office to epo]

Ref country code: IT

Effective date: 20080301